EP1572743B1 - Peptide und ihre verwendung zur behandlung von hiv infektionen - Google Patents

Peptide und ihre verwendung zur behandlung von hiv infektionen Download PDF

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EP1572743B1
EP1572743B1 EP03813584A EP03813584A EP1572743B1 EP 1572743 B1 EP1572743 B1 EP 1572743B1 EP 03813584 A EP03813584 A EP 03813584A EP 03813584 A EP03813584 A EP 03813584A EP 1572743 B1 EP1572743 B1 EP 1572743B1
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vir
seq
amino acid
peptides
cysteine
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French (fr)
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EP1572743A2 (de
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Knut Adermann
Frank Kirchhoff
Jan MÜNCH
Axel Schulz
Wolf-Georg Forssmann
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IPF Pharmaceuticals GmbH
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IPF Pharmaceuticals GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • the present invention relates to peptides which exhibit inhibitory activity on the infection of human cells by human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • Entry inhibitors block the uptake of HIV viral particles into blood cells by blocking one of the molecular steps occuring during viral entry.
  • An important step is binding of HIV to one of the major chemokine coreceptors CCR5 and CXCR4 (CC chemokine receptor 5 and CXCR chemokine receptor 4). These coreceptors are located on the surface of blood cells and are required to bind to HIV envelope proteins before viral entry.
  • Another step of viral interaction with cells required for fusion is the binding of the HIV envelope protein gp120 to cellular CD4 receptors. These steps are often referred to as attachment of the viral particle to cellular targets.
  • the blocking of the binding of HIV to chemokine coreceptors has been shown to suppress viral entry ( Strizki J.M., Proc. Natl.
  • the HIV protein gp41 has also been recognised as a potential target for anti-HIV drug development ( Gordon et al., AIDS Research and Human Retroviruses 11, 677-686, 1995 ).
  • the first approved fusion inhibitor is enfuvirtide (T-20, Fuzeon, DP178) ( WO 01/51673 A2 ; WO 96/40191 ; Cervia J.S et al., Clin. Infect.
  • HR-2 has not been shown to bind to protein segments other than HR-1 of HIV gp41 or even other molecules of viral or eukaryotic origin.
  • VIRIP virus inhibiting peptide
  • the problem remains unsolved that there is still no cure against AIDS, because the known therapeutics, though capable of significantly lowering the level of HIV in the body and of HIV-infected blood cells, do not remove the virus entirely.
  • a special drawback is, that the HIV is especially prone to mutations, which often result in the development of resistance against certain therapeutics.
  • the known therapeutics are only sufficiently effective if they are administered in combination with other therapeutics.
  • Such combined therapies at present extend the lifespan of the average patient without providing a cure, and are generally accompanied by severe side effects and frequently do not allow the patient to lead a "normal" life.
  • the present invention faces the problem to provide new therapeutics, which will overcome the problems as described above, and will allow an efficient therapy or will contribute to an efficient combination therapy.
  • WO-A-01/03640 discloses a peptide with the following amino acid sequence: Z1-LEAIPMSIPPEVKFNKPFVF-Z2 (VIRIP), in addition to its biologically active fragments and/or variants and/or derivatives, in particular, amidated, acetylated, sulphated, polyethylene glycol (PEG) modified, phosphorylated and/or glycosylated derivatives and to peptides which can be prepared by multiple synthesis and which have the biological activity of VIRIP.
  • peptides provided by the present invention which interact at least with the fusion peptide of HIV gp41.
  • the fusion peptide is the very amino-terminal part of gp41 consisting of about 30 amino acid residues.
  • the hydrophobic fusion peptide of gp41 serves as an anchor connecting the viral particle with the cellular host membrane ( Dimitrov A.S. et al., Biochemistry, 2003, 42, 14150-14158 ; Mobley et al., Biochim. Biophys. Acta, 1999, 1418, 1-18 ), and the peptides of the present invention interfere with the HIV cell fusion process, and thus prevent viral entry.
  • the peptides of the present invention are those with a biological activity against HIV infection, having amino acid sequence Z 1 -LE-X 1 -IP-X 2 -X 3 -X 4 -P-X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -K-X 11 -X 12 -X 13 -X 14 -X 15 -Z 2 , wherein X 1 is a lysine, alanine, or aspartic acid; X 2 is a cysteine, methionine or isoleucine; X 3 is a serine, cysteine, lysine or glycine; X 4 is an isoleucine, alanine, phenylalanine or cysteine; X 5 is a proline, D-proline or a substituted L-or D-proline; X 6 is a cysteine or glutamic acid; X 7 is an amino acid with a hydrophobic or an aromatic side chain
  • X 7 is phenylalanine, cysteine, valine, isoleucine, leucine, 3,3-diphenylalanine, 1- naphthylalanine, or p-fluorophenylalanine
  • X 8 is a phenylalanine, leucine, alanine, tryptophan, glycine, cysteine, D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
  • X 9 is a phenylalanine or D-1,2,3,4-
  • a further embodiment are peptides according to the invention with a biological activity against infection by HIV, having the amino acid sequence Z 1 -LE-X 1 -IP-X 1 -X 3 -X 4 -P-X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -K-X 11 -FVF-Z 2 , wherein X 1 is a lysine, alanine or aspartic acid; X 2 is a cysteine, methionine or isoleucine; X 3 is a serine, cysteine or glycine; X 4 is an isoleucine or cysteine; X 5 is a proline, D-proline or any substituted L- or D-proline; X 6 is a cysteine or glutamic acid; X 7 is a phenylalanine, cysteine, valine, isoleucine or 3,3-diphenylalanine; X 8 is a phenylalan
  • X 9 is a phenylalanine or D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
  • Z 1 is preferably NH 2 or a sequence of 1 to 3 amino acid residues
  • Z 2 is preferably COOH or a sequence of 1 to 3 amino acid residues.
  • peptides according the invention with a biological activity against infection by HIV, having the amino acid sequence Z 1 -LE-X 2 -IP-X 2 -X 3 -IP-X 5 -X 6 -X 7 -X 8 -F-X 10 -KPFVF-Z 2 , wherein X 1 is a lysine, alanine or aspartic acid; X 2 is a cysteine, methionine or isoleucine; X 3 is a serine or glycine; X 5 is a L-proline, D-proline or any substituted L- or D-proline X 6 is a cysteine or glutamic acid; X 7 is a phenyalanine or valine; X 8 is a phenylalanine, leucine, alanine or L-1,2,3,4-tetrahydroIsoquinoline-3-carboxylic acid; X 10 is a glycine or asparag
  • Z 1 is preferably NH 2 or a sequence of 1 to 3 amino acid residues and Z 2 is preferably COOH or a sequence of 1 to 3 amino acid residues.
  • the biological activity against HIV infection of the peptide, as measured as IC 50 is equal of or below of 800 nM.
  • peptides of the invention with biological activity against infection by HIV having the amino acid sequence Z 1 -LEAIP-X 2 -SIP-X 5 -X 6 -V-X 8 -FNKPFVF-Z 2 , wherein X 2 and X 6 are cysteines, or X 2 is methionine and X 6 is glutamic acid X 5 is a D-proline or L-proline; X 8 is an amino acid with a hydrophobic or an aromatic side chain or lysine; Z 1 is NH 2 or a sequence of 1 to 10 amino acid residues; Z 2 is COOH or a sequence of 1 to 10 amino acid residues; and peptides which are covalently linked oligomers and/or amidated, alkylated, acylated, sulfated, pegylated, phosphorylated and/or glycosylated derivatives with the proviso that at least one of the following is true:
  • Z 1 is preferably NH 2 or a sequence of 1 to 3 amino acid residues and Z 2 is preferably COOH or a sequence of 1 to 3 amino acid residues.
  • an embodiment of the peptides of the present invention are those, wherein the cysteine residues at positions 6 and 11, 6 and 12, 7 and 12, or 8 and 13 are connected by an intramolecular disulfide bond.
  • the peptides with cysteine residues at these positions may occur with an Intramolecular bridge between these residues, or, under reductive conditions as linear molecules.
  • a further embodiment are peptides with a single cysteine residue, wherein said cysteine residue is connected by an inter-molecular disulfide bond to another peptide molecule with a single cysteine residue, forming a homo-dimer.
  • peptides wherein the leucine residue at amino acid position 1 and the glutamic acid at amino acid position 2 are covalently linked by an N-alkylated amide bond or by an ester bond or by a reduced peptide bond or by a retro-inverso peptide bond or by an N-alkylated retro-inverso peptide bond.
  • a further embodiment are peptides which interact with the HIV fusion peptide of gp41.
  • the peptides of the present invention are characterised by an IC 50 of equal or below 6500 nM, preferably an IC 50 of equal or below 2000 nM and most preferably an IC 50 of equal or below 800 nM, such as VIR-344 (SEQ ID NO.
  • VIR-345 SEQ ID NO. 50
  • VIR-353 SEQ ID NO. 56
  • VIR-357 SEQ ID NO. 60
  • VIR-358 SEQ ID NO. 61
  • VIR-449 SEQ ID NO 73
  • VIR-455 SEQ ID NO 76
  • VIR-484 SEQ ID NO 79
  • VIR-576 SEQ ID NO: 86
  • VIR-580 SEQ ID NO. 87
  • nucleic acids coding for these peptides are embodiments of the present invention. Further embodiments are antibodies binding specifically to these peptides.
  • a further embodiment is a medicament containing anyone of these peptides, nucleic acids coding for these peptides, or specific antibodies directed against these peptides.
  • the medicament is in galenic formulations for oral, intravenous, intramuscular, intracutaneous, subcutaneous, intrathecal administration, and as an aerosol for transpulmonary administration.
  • a further embodiment is said medicament comprising at least one further therapeutic agent.
  • an embodiment is the medicament, wherein the said at least one further therapeutic agent is a viral protease inhibitor, a reverse transcriptase inhibitor, a fusion inhibitor, a cytokine, a cytokine inhibitor, a glycosylation inhibitor or a viral mRNA inhibitor, etc.
  • a viral protease inhibitor a reverse transcriptase inhibitor
  • a fusion inhibitor a cytokine
  • a cytokine inhibitor a glycosylation inhibitor or a viral mRNA inhibitor, etc.
  • an assay for determining molecules capable of interacting with the fusion peptide of HIV comprising anyone of the above peptides of the invention. Use of these peptides in said assay is also an embodiment.
  • a further embodiment is a diagnostic agent containing these peptides, nucleic acids or antibodies.
  • One more embodiment is use of the diagnostic agent for assay systems for testing isolated plasma, tissue, urine and cerebrospinal fluid levels for HIV infection. Further specific embodiments of the present invention are the
  • the peptides of the present invention are related to the hemofiltrate-derived peptide VIRIP (SEQ ID No. 1) as disclosed and described in WO 01/34640 , which has biological activity in preventing infection by HIV. They differ from VIRIP e.g. in amino acid position 13, where VIRIP contains a lysine residue, while the peptides of the present invention do not contain a lysine residue at amino acid position 13 or have further amino acid changes throughout their 21 amino acids in comparison to VIRIP.
  • the peptides of the present invention all posses significantly higher anti-HIV activity (measured as IC 50 against two HIV-1 strains) than VIRIP.
  • the increase in anti-HIV activity is at least 4-fold (VIR-184, SEQ ID NO. 12), and the very active peptides of the present invention are up to 161-fold (e.g. VIR-280, SEQ ID NO. 39) more active against HIV than VIRIP.
  • the peptides of the invention are based on an amino acid sequence of 21 amino acids, with possible extensions of 1 to 10 amino acids at both ends according to Z 1 and Z 2 , whereby an extension of 3 amino acids is preferred.
  • the amino acid numbering used herein always corresponds to the amino acids 1 to 21 of the basic sequence irrespective of a possible N-terminal extension due to a residue Z 1 , such that amino acid position 1 corresponds to leucine and amino acid position 21 to phenylalanine or a deletion.
  • the common amino acid one and three letter codes are used. If not indicated otherwise, the L-enantiomers of amino acids were used.
  • the small letter “p” stands for D-proline. Other D-enantiomers are indicated by a "D-" prefix.
  • Tic stands for tetrahydrisioquinoline carboxylic acid.
  • Oic stands for octahydroindole carboxylic acid.
  • hydrophobic amino acid as used herein is readily understood by the skilled person. In particular, it refers to any of the amino acids glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, and non-endogenous hydrophobic amino acids.
  • aromatic amino acid as used herein is readily understood by the skilled person. In particular it refers to any of the amino acids phenylalanine, tyrosine, tryptophan, histidine, and non-endogenous aromatic amino acids, such as 1-naphthylalanine, 3,3-diphenylalanine, p-fluorophenylalanine, or D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, etc.
  • covalently linked oligomers is readily understood by the skilled person. In particular it refers to multiple peptide chains covalently linked to each other.
  • the peptide chains can have the identical or a different amino acid sequence.
  • the covalent bond can be a direct bond between the respective peptide chains such as a disulfide bond, thioether bond, ether bond, amide bond.
  • the peptide chains can also be covalently linked by a spacer of any chemical nature ( Houben-Weyl, Methods of organic chemistry, Synthesis of peptides and peptidomimetics, Georg Thieme Verlag, Stuttgart 2002 ).
  • derivative is readily understood by the skilled person. In particular it refers to a chemically modified peptide. This modification could be chemical modifications of the peptide at the N- and C-terminus, the side chains of the peptide, the C ⁇ - and N ⁇ -atoms of the peptide backbone, and the atoms forming the peptide bonds of the backbone.
  • acylated is readily understood by the skilled person. In particular it refers to peptides that contain a covalently linked carboxylic acid residue other than an amino acid at amino groups at the N-terminus and/or at side chains of amino groups.
  • alkylated is readily understood by the skilled person. In particular it refers to peptides which are modified with an alkyl group of various length and structure at the N-terminal amino group, at any backbone atom and/or at any functional group of a side chain.
  • sulfated is readily understood by the skilled person. In particular it refers to peptides carrying a sulfate moiety at the hydroxyl group of a tyrosine or substituted tyrosine derivative residue.
  • pegylated is readily understood by the skilled person. In particular it refers to peptides which contain covalently linked a polyethyleneglycol (PEG) moiety consisting of at least two repeating units -CH 2 -CH 2 -O- typical of polyethyleneglycol. Preferred is a so-called mini-PEG group. Pegyl groups may have a molecular weight of up to 20 KDa and can be bound to different functional groups in a peptide sequence directly or via a spacer group at the N- and/or C-terminus and/or side chain functional groups.
  • PEG polyethyleneglycol
  • the spacer group is selected from the group of bifunctional hydrocarbon chains characterised by a backbone of two, three, four, five, six, seven, eight or nine carbon atoms, and two functional groups, such as two amino groups, two carboxyl groups or one amino group and one carboxyl group.
  • One or more pegyl groups can be contained at different sites of a peptide.
  • phosphorylated is readily understood by the skilled person. In particular it refers to peptides where the hydroxy groups of the side chains of threonine, serine, hydroproline, hydroxylysine, tyrosine, and/or any other non-natural hydroxy amino acid is esterified with a phosphate group.
  • glycosylated is readily understood by the skilled person. In particular it refers to peptides that contain a monomeric and/or oligomeric carbohydrate moiety which is linked via the glycosilic or an alcoholic hydroxy group to the side chains of serine, threonine, tyrosine, asparagine, and/or non-natural amino acids.
  • cyclic is readily understood by the skilled person. In particular it refers to peptides that contain a cyclic structural motif.
  • the cyclization can be achieved by backbone cyclization or by linking a side chain of an amino acid to a side chain of a different amino acid present in the same molecule.
  • two cysteine residues of a peptide or one carboxylic acid side chain and one amino group-containing side chain form a cyclic motif via a disulfide bond or an amide bond.
  • VIR-165 SEQ ID NO. 7
  • VIR-166 SEQ ID NO. 8
  • VIR-272 SEQ ID NO. 36
  • VIR-273 SEQ ID NO. 37
  • VIR-274 SEQ ID NO. 38
  • VIR-280 SEQ ID NO. 39
  • VIR-344 SEQ ID NO. 49
  • VIR-345 SEQ ID NO. 50
  • VIR-346 SEQ ID NO. 51
  • VIR-348 SEQ ID NO. 52
  • VIR-350 SEQ ID NO. 53
  • VIR-351 SEQ ID NO. 54
  • VIR-352 SEQ ID NO. 55
  • VIR-353 SEQ ID NO. 56
  • VIR-354 SEQ ID NO.
  • VIR-355 SEQ ID NO. 58
  • VIR-356 SEQ ID NO. 59
  • VIR-357 SEQ ID NO. 60
  • VIR-358 SEQ ID NO. 61
  • VIR-568 SEQ ID NO. 84
  • VIR-570 SEQ ID NO. 85
  • VIR-576 SEQ ID NO. 86
  • VIRIP SEQ ID NO. 1
  • peptides with a significantly increased activity against HIV were obtained.
  • the most significant increase in activity is observed, when the L-proline at position 10 is substituted by a D-proline, and/or two cysteines are introduced at amino acid positions 6 and 11, and/or when the positively charged lysine at position 13 is exchanged against an amino acid with a hydrophobic or aromatic side chain. It is believed that the activity when compared to wild-type VIRIP (SEQ ID NO. 1) is increased due to a change in structure.
  • Cysteine bridges are known to alter the structure and to reduce the flexibility of a peptide significantly, as well as the introduction of a D-proline, which causes a change in secondary structural elements of a peptide and thus a changed orientation of different parts of the peptide to each other. Furthermore, the exchange of a lysine against an uncharged hydrophobic or aromatic amino acid will alter the structure, because a possible interaction of the positively charged lysine side chain with the negatively charged amino acids at positions 2 and 11 of the same molecule, or with a negatively charged portion of a receptor molecule is changed.
  • a significant increase in the anti-HIV activity is further observed when the alanine residue at position 3 is exchanged to a positively or negatively charge residue by substitution with lysine or aspartic acid residues.
  • the introduction of a charged residue at position 3 can enhance the binding strength to a corresponding part of a receptor molecule by increased electrostatic or dipolar forces.
  • the exchange of the amino acid residues at positions 7 or/and 15 against a small amino acid residue, in particular glycine has also been found to increase the anti-HIV activity.
  • Glycine residues are the least sterically hindering residues and allow an optimal internal structural arrangement of a peptide when binding to a receptor molecule or when forming aggregates with themselves required for binding with a receptor molecule.
  • a dimerization of peptides of the invention can be achieved chemically by covalent linking of two identical peptide chains.
  • the covalent link can be a direct bond between side chain functional groups such as the thiol group of cysteine residues, or a bond involving a spacer between the peptide chains as is present when two identical chains of a peptide of the invention are bound to the two amino groups of a lysine residue.
  • the latter is often referred to as the smallest form of a lysine-core dendrimer ( Sadler K., J.
  • Oligomers in particular dimers of peptides of the invention, can induce a structurally and/or biologically more stable form of the molecules. In addition, they can increase the local concentration of the antivirally active peptide at the site of action. They can thus provide forms of the peptides of the invention which interact more favourable with a receptor molecule.
  • Peptides according to the invention can be easily chemically synthesised or produced by recombinant expression. Due to the small size, i.e. the low number of amino acid the peptides of the invention are composed of, the entire peptide synthesis technologies can be utilised to chemically synthesise such substances. In comparison to the synthesis of the HIV fusion inhibitor T-20, which requires the synthesis of three individual fragments, and subsequently the joining of the three fragments to give rise to the final product T-20, the peptides of the present invention, can be synthesised at large scale by stepwise solid phase methods or by solution phase chemistry. Thus the manufacturing process of the peptides of the present invention is straightforward and therefore the costs of the goods comprising the peptides of the present invention are lower. A further advantage of the peptides of the present invention is their solubility and stability over a broad range of pH (pH 2 - 8.5) in solvents of different ionic strength.
  • the chemical synthesis can be carried out on a solid support using solid-phase technologies or in solution phase, both being standard methods known to the skilled person.
  • Peptides according to the invention can also be synthesized by the ligation of two or more side chain-protected or side chain-unprotected fragments, standard methods known to the skilled person ( Tam J.P., Biopolymers, 2001, 60, 194-205 ).
  • the solid-phase synthesis of peptides according to the invention or its fragments can be carried out using the Fmoc/tBu- or Boc/Bzl-protection pattern of amino acids. Other protective groups that are not in the standard Fmoc-protection scheme can be used.
  • a disulfide bond into peptides of the invention may be achieved by applying oxidative chemical methods with peptides containing two cysteine residues known to the skilled person ( Pennington et al. (editors), Peptide synthesis protocols, Humana Press, Totowa 1994 ; Chan W.C. et al. (editors), Fmoc solid phase peptide synthesis: A practical approach, Oxford University Press, Oxford, 2000 ).
  • Disulfides of peptides of the invention may be generated from reduced precursor peptides containing one or two unprotected cysteine residues obtained from solid-phase or solution synthesis by oxidative treatment.
  • Oxidizing agents oxygen, dimethylsulfoxide, iron(III) salts, iodine, or others may be used.
  • Disulfides of peptides of the invention may alternatively be introduced into the peptides from precursors containing protective groups at the corresponding cysteine residues.
  • protective groups acetamidomethyl, tert-butyl, S-tert-butyl or others may be used.
  • Cleavage of protective groups and intra-chain disulfide bond formation may be carried out using agents such as iodine, phosphines, or others.
  • Cyclic peptides other than those with a disulfide bond can be obtained via backbone cyclization of the peptide or via a chemical bond between at least one reactive side chain group such as amino, carboxy, hydroxy or thio and any other reactive group present in the same molecule, as known to the skilled person ( Li et al., Curr. Top. Med. Chem., 2002, 2, 325-341 ; Tam J.P., Biopolymers, 2001, 60, 194-205 ; Goodman M., Houben-Weyl, Methods of organic chemistry, Synthesis of peptides and peptidomimetics, Georg Thieme Verlag, Stuttgart 2002 ).
  • Covalently linked oligomers of peptides are obtained by linking two peptide chains via different types of chemical bonds.
  • Disulfide-linked oligomers are synthesized by coupling the two peptide chains either via activated cysteines or without any preactivation of the cysteines ( Sacca B. et al., J. Pept. Sci., 2002, 8, 192-204 ; Seewald N. et al., Peptides: biology and chemistry, Wiley-VCH, Weinheim, 2002 ).
  • Thioether bonds and ether bonds and peptide bonds between two peptide chains can be introduced according to different methods known to the skilled person and described in the literature ( Seewald N.
  • Lysine-core dendrimers can be synthesized by coupling Fmoc-Lys(Fmoc)-OH to a solid support. After deprotection of the amino acid solid phase peptide synthesis leads to the oligomeric peptides ( Seewald N. et al., Peptides: biology and chemistry, Wiley-VCH, Weinheim, 2002 ; Chan W.C. (editors) Fmoc solid phase peptide synthesis: A practical approach, Oxford University Press, Oxford 2000 ). Lysine can be replaced by any other amino acid containing two amino groups.
  • Amidated peptides are obtained by solid phase peptide synthesis using resins carrying an amide linker on which the peptide chain is assembled. Acid cleavage of correspondingly synthesized peptides results in peptide amides. In solution phase synthesis amidated peptides are obtained when the C-terminal amino acid is used as a building block which has a preformed carboxamide at the C-terminus. ( Chan W.C. (editors) Fmoc solid phase peptide synthesis: A practical approach, Oxford University Press, Oxford 2000 ).
  • Acylated peptides are obtained by the skilled person through converting a peptide with free amino or hydroxy groups using activated acylation reagents derived from carboxylic acids such as acyl halogenides or carboxylic anhydride or other reactive carbonyl compounds to a corresponding acylated peptide.
  • carboxylic acids such as acyl halogenides or carboxylic anhydride or other reactive carbonyl compounds
  • Acylated peptides are obtained by the skilled person through converting a peptide with free amino or hydroxy groups using activated acylation reagents derived from carboxylic acids such as acyl halogenides or carboxylic anhydride or other reactive carbonyl compounds to a corresponding acylated peptide.
  • acylation reagents derived from carboxylic acids such as acyl halogenides or carboxylic anhydride or other reactive carbonyl compounds
  • a acylated peptide can be achieved using free carboxylic acids
  • Alkylated peptides may be obtained by incorporating prealkylated amino acid building blocks when carrying out peptide synthesis on the solid support or in solution. Such amino acids are coupled onto the peptide chains using standard activation protocols known to the skilled person ( Chan W.C. (editors) Fmoc solid phase peptide synthesis: A practical approach, Oxford University Press, Oxford 2000 ). Alkylation may also be achieved after assembly of a peptide chain by using appropriate alkylation methods known to the skilled person ( Greene T.W., Protective groups in organic chemistry, John Wiley & Sons, New York, 1991 ; Kocienski P., Protecting groups, Thieme-Verlag, Stuttgart 1994 ).
  • Such methods may be applied to reactive groups such as amino, hydroxy, thio and peptide bonds of the peptide backbone in a partially protected peptide.
  • Sulfated peptides are obtained by using presulfated building blocks of tyrosine or tyrosine derivatives in solid phase or solution peptide synthesis. O-sulfates remain attached to the hydroxy group during peptide cleavage from the resin when highly acid-labile resins such as 2-chlorotrityl resin are used for synthesis ( Seewald N. et al., Peptides: biology and chemistry, Wiley-VCH, Weinheim, 2002 ).
  • Pegylated peptides contain pegyl residues bound to functional groups of a peptide.
  • Pegyl residues are characterized as hydrophilic linear or branched polymeric chains with a repeating unit -CH 2 -CH 2 O-.
  • Pegyl residues are introduced into a peptide after assembly of the peptide chain using suitable functionally modified and reactive pegyl-containing substances.
  • Various activated pegyl groups can be attached by the skilled person to peptides by different activation methods to different side chains or terminal functional groups of a peptide such as amino, carboxyl, hydroxy and thio ( Veronese F.M. et al., Bioconjug. Chem., 2001, 12, 62-70 ; Veronese F.M., Biomaterials, 2001, 22, 405-417 ).
  • Phosphorylated peptides can be synthesized by solid phase or solution phase peptide synthesis. Synthesis of phosphorylated peptides is usually achieved by the skilled person utilizing phosphorylated hydroxy amino acid building blocks and/or by post-chain assembly phosphorylation of protected peptides with one or more free hydroxy functional groups ( Murray J.S., Biopolymers; 2001, 60, 3-31 ; Chan W.C. et al. (editors), Fmoc solid phase peptide synthesis: A practical approach, Oxford University Press, Oxford, 2000 ; Seewald N. et al., Peptides: biology and chemistry, Wiley-VCH, Weinheim, 2002 ).
  • Glycosylated peptides can be obtained by the skilled person using glycosylated amino acid building blocks which can be incorporated into soild phase or solution phase synthesis of peptides or by the global post-chain assembly glycosylation approach ( Davis B.G., Chem. Rev., 2002, 102, 579-602 ; Chan W.C. et al. (editors), Fmoc solid phase peptide synthesis: A practical approach, Oxford University Press, Oxford, 2000 ; Seewald N. et al., Peptides: biology and chemistry, Wiley-VCH, Weinheim, 2002 ).
  • the invention also relates to nucleic acids coding for peptides of the invention.
  • Preferred nucleic acids are DNA and RNA, especially cDNA and mRNA.
  • Subject of the invention are also antibodies specifically binding to peptides of the invention.
  • the term "specifically” is readily understood by the skilled person. In particular, it means that the antibodies do not bind or do essentially not bind related peptides like VIRIP which are not peptides of the invention.
  • a person skilled in the art obtains antibodies against peptides of the invention by routine methods, and will select specific antibodies of the invention by known screening methods.
  • the invention relates to peptides which specifically interact with and bind to the N-terminal region of the envelope protein gp41 of HIV.
  • the term "interact with” and “bind to” is readily understood by the skilled person. By such binding and interaction, peptides of the invention block infection of host cells by HIV particles.
  • the present invention also relates to peptides which bind to synthetic peptides corresponding to the fusion peptide of gp41 of HIV.
  • a person skilled in the art detects binding and interaction of peptides of the invention to the synthetic fusion peptide of gp41 of HIV by applying quantitative structure/activity relationship (QSAR) assays.
  • QSAR quantitative structure/activity relationship
  • These assays comprise but are not limited to the detection of the suppression of the hemolytic effect of the synthetic fusion peptide ( Mobley P.W. et al., Biochim. Biophys. Acta, 1992, 1139, 251-256 ; Gordon L., Biochim. Biophys. Acta, 1992, 1139, 257-274 ), microcalorimetry ( Gohlke H. et al., Angew. Chem. Int. Ed. Engl., 2002, 41, 2644-2676 ), or NMR-spectroscopical techniques which can be chemical shift titration experiments or saturation transfer difference spectroscopy ( Meyer et al., Ernst Schering Res. Found. Workshop, 2004, 44, 149-167 ).
  • the invention also relates to a medicament containing the peptides, nucleic acids or antibodies of the invention.
  • the medicament is preferably provided in galenic formulations for oral, intravenous, intramuscular, intracutaneous, subcutaneous, intrathecal administration, or as an aerosol for transpulmonary administration.
  • the medicament comprises at least one further therapeutic agent.
  • Said at least one further therapeutic agent can be a viral protease inhibitor, a reverse transcriptase inhibitor, a fusion inhibitor, a cytokine, a cytokine inhibitor, a glycosylation inhibitor or a viral mRNA inhibitor, etc.
  • such inhibitors are directed against HIV.
  • Such combined therapeutics are highly relevant in the treatment of AIDS.
  • the peptides, nucleic acids and antibodies of the invention are preferably used in manufacturing of a medicament for the treatment of HIV infections. This comprises all known strains of the retrovirus HIV (human immunodeficiency virus), especially the most common strains of HIV-1. HIV-1 is associated with the outbreak of AIDS.
  • the invention also relates to a diagnostic agent containing peptides, nucleic acids or antibodies of the invention.
  • the diagnostic agent may be used for assay systems for testing isolated plasma, serum, tissue, urine and cerebrospinal fluid levels for HIV infections.
  • the invention also relates to assay systems which involve peptides of the invention as a tool to identify substances which bind to the envelope protein gp41 of HIV, in particular the N-terminal fusion peptide of gp41.
  • assays can be any system which is suitable to measure the binding of any substance to the fusion peptide either integrated in the entire gp41 protein in isolated, viral, or any other form, or in synthetic form with a length up to 35 amino acid residues starting with the very N-terminus of gp41.
  • assays which can be any spectroscopical, cellular, or radio-ligand assay, the binding of a substance in competition to peptides of the invention is measured.
  • VIR-161 SEQ ID NO. 3
  • VIR-162 SEQ ID NO. 4
  • VIR-163 SEQ ID NO. 5
  • VIR-164 SEQ ID NO. 6
  • VIR-165 SEQ ID NO. 7
  • VIR-166 SEQ ID NO. 8
  • VIR-170 SEQ ID NO. 9
  • VIR-175 SEQ ID NO. 10
  • VIR-182 SEQ ID NO. 11
  • VIR-184 SEQ ID NO. 12
  • VIR-190 SEQ ID NO. 13
  • VIR-191 SEQ ID NO. 14
  • VIR-192 SEQ ID NO.
  • VIR-193 SEQ ID NO. 16
  • VIR-197 SEQ ID NO. 17
  • VIR-199 SEQ ID NO. 18
  • VIR-229 SEQ ID NO. 19
  • VIR-234 SEQ ID NO. 20
  • VIR-243 SEQ ID NO. 21
  • VIR-252 SEQ ID NO. 22
  • VIR-255 SEQ ID NO. 23
  • VIR-257 SEQ ID NO. 24
  • VIR-258 SEQ ID NO. 25
  • VIR-259 SEQ ID NO. 26
  • VIR-260 SEQ ID NO. 27
  • VIR-261 SEQ ID NO. 28
  • VIR-262 SEQ ID NO. 29
  • VIR-263 SEQ ID NO. 30
  • VIR-264 SEQ ID NO.
  • VIR-265 SEQ ID NO. 32
  • VIR-266 SEQ ID NO. 33
  • VIR-268 SEQ ID NO. 34
  • VIR-269 SEQ ID NO. 35
  • the second set of experiments concerned the efficacy of the peptides of the present invention to inhibit HIV infection (see table 2).
  • the peptides were tested on two HIV-1 strains and IC 50 values were calculated.
  • the most active peptides had an IC 50 of equal or below 800 nM, whereby for example VIR-484 (SEQ ID NO. 79) had an IC 50 of 100 nM.
  • Peptides with still considerable activity where those with an IC 50 of equal or below 2000 nM, and those with an IC 50 of equal or below 6500 nM still had an increased activity in comparison to the native VIRIP (SEQ ID NO. 1); the native VIRIP (SEQ ID NO.
  • the third set of experiments determined the in vivo toxicity of the VIRIP peptides of the present invention. Considering the positive outcome of the in vitro cytotoxicity test, it was sufficient to test only one compound. Mice were injected with VIR-121 (SEQ ID NO. 2), observed over a period before sacrificing them. Throughout the life of the mice no signs of reduced or increased motility, dyspnea, ataxia, nor a reduced or increased muscle tone were observed. No changes of behaviour were observed, and behaviour was comparable to that of the control animals. The pathological examination did not reveal any abnormalities. It was therefore concluded that the peptides of the present invention are well tolerated by a living organism.
  • a forth set of experiments concerned the stability of the peptides of the present invention in mammalian plasma (see table 3).
  • Plasma isolated from various animals and humans was spiked with defined amounts of various peptides of the present invention.
  • the peptides displayed a considerable half life in human plasma, most prominent being VIR-512 (SEQ ID NO. 83), VIR-580 (SEQ ID NO. 87) and VIR-357 (SEQ ID NO. 60), with a half-life of 315 h, 38.9 h and 23.3 h, respectively.
  • VIR-512 SEQ ID NO. 83
  • VIR-580 SEQ ID NO. 87
  • VIR-357 SEQ ID NO. 60
  • These peptides also showed considerable stability in the animal plasma, but the actual values varied from those found for human plasma.
  • the native VIRIP (SEQ ID NO. 1) has a half-life of 53.7 h in human plasma.
  • the results also showed, that rat plasma is not a suitable model system for these type
  • the peptides of the present invention are characterised by their anti-HIV activity, which, expressed as IC 50 , is equal to or below 6500 nM, whereby the most active peptides have an IC 50 of below 800 nM. Individual peptides of the present invention were found to have IC 50 of below 100 nM (see table 1).
  • Example 1 Chemical synthesis of peptides of the present invention
  • the peptides according to the invention were chemically synthesized utilizing the principle of solid-phase peptide synthesis and the Fmoc or Boc protective group strategy ( Atherton and Sheppard, 1989, Solid Phase Peptide Synthesis, IRL Press ; Merrifield, 1986, Solid phase synthesis, Science 232, 341-347 ), but can also be synthesized with solution phase synthesis or by coupling protected or unprotected fragments of the peptides according to the invention.
  • peptide VIR-199 amino acid sequence: LEAIPMSIPpEFLFNKPFVF
  • SEQ ID NO. 18 fluorenylmethoxycarbonyl (Fmoc)-protected amino acids on an automated peptide synthesizer 433A (Applied Biosystems).
  • the synthesis was performed using a preloaded Fmoc-Phe-Wang resin with a loading capacity of 1 mmol/g resin with standard HBTU [(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate)/HOBt (1-hydroxybenzotriazol) activation with capping cycles using acetic anhydride in N-methylpyrrolidinone (NMP) at a scale of 0.2 mmol.
  • NMP N-methylpyrrolidinone
  • the resulting precipitate was separated by centrifugation, washed with TBME and dried under vacuum.
  • the crude peptide was dissolved in diluted acetic acid and loaded onto a preparative Vydac C18 column (47x300 mm, 15-20 ⁇ m, flow rate 40 ml/min; solvent A, 0.07 volume % TFA; solvent B, 0.07 volume % TFA in acetonitrile/H 2 O 80:20 (volume %); UV detection at 215 nm; with the following gradient: 45-70 volume % B in 50 min.
  • the yield of the peptide LEAIPMSIPpEFLFNKPFVF SEQ ID NO. 18 was 138 mg.
  • the process for synthesis of the peptides according to the invention was adapted to larger scales ranging from 0.5 to 20 mmol yielding purified peptides of the present invention in amounts between 1 g and 5 g.
  • the synthesis process was also adapted to small-scale multiple peptide synthesis.
  • Peptides according to the invention having intramolecular disulfide bonds were treated with air at pH 7.5-8.5, with or without dimethylsulfoxide, or alternatively, from linear precursors with two acetamidomethyl-protected cysteine residues by iodine oxidation to facilitate cysteine bridge formation.
  • Example 2 Cytotoxicity of the peptides of present invention on human cells
  • THP-1 cells The cytotoxicity of peptides of the invention was tested by evaluating the viability of human monocytic THP-1 cells. Cytotoxic effects of the peptides were tested by their influence on metabolic activity by means of the WST-1 assay (Roche Diagnostics, Germany). THP-1 cells were incubated with test peptides in a 96-well plate (approx. 25,000 cells per well) for 24 hours in RPMI-1640 medium containing 25 mM L-glutamine and 10 volume % fetal calf serum at 37 °C in an atmosphere with 5 volume % CO 2 , Ten ⁇ l of a WST-1 solution was added to each cavity, and incubation of THP-1 cells was allowed for 2 further hours at corresponding conditions.
  • Metabolically active THP-1 cells reduce WST-1, a light red tetrazolium salt, yielding a soluble yellow formazan salt.
  • the known cytotoxic substance cycloheximide was used at a concentration of 50 ⁇ g/ml; the cytotoxicity of cycloheximide was set to 100%.
  • the peptide MBI-28 a highly cytotoxic peptide known to the skilled person, was used with a maximum concentration of 300 ⁇ g/mL.
  • V ⁇ i ⁇ a ⁇ b ⁇ i ⁇ l ⁇ i ⁇ t ⁇ y % A 450 ⁇ n ⁇ m p ⁇ e ⁇ p ⁇ t ⁇ i ⁇ d ⁇ e - A 450 ⁇ n ⁇ m c ⁇ y ⁇ c ⁇ l ⁇ o ⁇ h ⁇ e ⁇ x ⁇ i ⁇ m ⁇ i d ⁇ e / A 450 ⁇ n ⁇ m n ⁇ e ⁇ g ⁇ a ⁇ t ⁇ i ⁇ v ⁇ e ⁇ c ⁇ o ⁇ n ⁇ t ⁇ r ⁇ o ⁇ l
  • VIR-191 SEQ ID NO. 14
  • VIR-192 SEQ ID NO. 15
  • VIR-193 SEQ ID NO. 16
  • VIR-197 SEQ ID NO. 17
  • VIR-199 SEQ ID NO. 18
  • VIR-229 SEQ ID NO. 19
  • VIR-234 SEQ ID NO. 20
  • VIR-243 SEQ ID NO. 21
  • VIR-252 SEQ ID NO. 22
  • VIR-255 SEQ ID NO. 23
  • VIR-257 SEQ ID NO. 24
  • VIR-258 SEQ ID NO. 25
  • VIR-259 SEQ ID NO. 26
  • VIR-260 SEQ ID NO. 27
  • VIR-261 SEQ ID NO. 28
  • VIR-262 SEQ ID NO.
  • VIR-263 SEQ ID NO. 30
  • VIR-264 SEQ ID NO. 31
  • VIR-265 SEQ ID NO. 32
  • VIR-266 SEQ ID NO. 33
  • VIR-268 SEQ ID NO. 34
  • VIR-269 SEQ ID NO. 35
  • Example 3 Inhibition of the HIV infection by the peptides of present invention
  • P4-CCR5 indicator cells ( Charneau et al., 1994; Journal of Molecular Biology 241, 651-662 ) expressing the primary CD4 receptor and both major HIV-1 entry cofactors CXCR4 and CCR5, were used to evaluate whether peptides according to the invention are potent inhibitors of HIV-1 infection.
  • These cells contain the ß-galactosidase reporter gene under the control of the HIV-1 promoter.
  • activation of the ß-galactosidase reporter gene allows to measure the efficiency of HIV-1 infection and thus to quantitate the potency of HIV-1 inhibitors ( Detheux M. et al., 2000; Journal of Experimental Medicine 192, 1501-1508 ; Münch et al., 2002; Antimicrobial Agents and Chemotherapy 46, 982-990 ).
  • P4-CCR5 cells ( Charneau et al., 1994; Journal of Molecular Biology 241, 651-662 ; Charneau et al., Virology. 1994 205, 247-53 ) were kept in RPMI 1640 medium supplemented with 10 volume % FCS. This cell line coexpresses CD4 and both HIV-1 coreceptors CCR5 and CXCR4 and contains the ß-galactosidase gene under the control of the HIV-1 promoter. Virus stocks were generated by the calcium coprecipitation method as described ( Detheux et al., J Exp Med.
  • peptides according to the invention have greatly enhanced anti-HIV-1 activity as compared to VIRIP.
  • Peptides of the invention inhibited the infection by the X4-tropic HIV-1 NL4-3 and the HIV-1 NL4-3 DTV (from hereon called DTV) - DTV is a variant of NL4-3 and was originally described by Rimsky et al. (Journal of Virology 72, 986-993; 1998 ) as r4 - molecular clones with more than 10-fold up to more than 100-fold higher efficiency than the original VIRIP.
  • Peptides of the invention were also active against infection by the R5-tropic HIV-1 YU-2 molecular clone.
  • Example 4 Toxicity of the peptides of the present invention in mice
  • VIR-121 LMAIPMSIPpEVAFNKPFVF
  • Three animals were treated with the test substance, and the animals were observed at time points of 5, 15, 30 min, and 1, 3, 6, and 24 hours after administration of the sample into the tail vein. As a control, 3 mice were each treated with a corresponding volume of vehicle (0.9 volume % NaCl).
  • Example 5 Stability of peptides of the invention in mammalian plasma
  • peptides of the invention were examined in mammalian plasma after incubation in EDTA plasma obtained from human, dog, cynomolgus and rat at 37 °C. Plasma was spiked resulting in concentrations of 40 ⁇ g/ml and stored at 37 °C. At time points 0, 15, 30, 45, 60, 120, 180, 240 and 300 min samples of 20 ⁇ l were taken. The plasma was immediately mixed for precipitation with the two-fold volume of acetonitrile containing 0.15% (w/v) n- nonyl- ⁇ -D-glucopyranoside.
  • eluent A water containing 0.06% trifluroacetic acid (v/v)
  • eluent B acetonitrile/water 80:20 (v/v; with 0.05% trifluoroacetic acid; v/v).
  • a C18 precolumn was used in combination with a C18 separation column (300 ⁇ , 5 ⁇ m, 150 x 1 mm inner diameter) at a flow rate of 30 ⁇ l/min.
  • HPLC eluates were ionized by the electrospray technique of a LCQ classic mass spectrometer. Areas of the detected peaks of the peptides of te present invention were measured and used for quantification by external calibration.
  • the calibration curve was linear over a range from 0.5 ⁇ g/ml to 250 ⁇ g/ml plasma.
  • Half life - defined as the period for a concentration decrease to 50% of the initial concentration - was calculated from the slope of an extrapolated curve plotting the relative peptide concentration at a given time point (logarithm scale) against the incubation time.
  • the synthetic fusion peptide of HIV gp41 causes concentration-dependent hemolysis which can be measured by hemoglobin released by erythrocytes. Peptides and any other substance binding to the fusion peptide impair its potency to lyse erythrocytes by changing its structural properties.
  • the inhibition of fusion peptide induced hemolysis was tested as follows: Blood from healthy donors was collected in citrate monovettes and the erythrocytes were extracted by a standard centrifugation and washing protocol known to the skilled person. The final erythrocyte-containing pellett was diluted 1:100 with phosphate-saline buffer.
  • the 96-well plate is centrifuged 5 min at 2800 rpm and of the supernatant fluid 150 ⁇ l were transferred to a flat-bottom microtiter plate, and the absorbance was measured at 450 nm.
  • the percentage hemolysis was calculated by: [(A450 of the peptide treated sample - A450 of buffer treated sample)/(A450 of Tween-20 treated sample - A450 of buffer treated sample)] x 100%.
  • results show that the fusion peptide-induced hemolysis is inhibited upon addition of increasing concentrations of peptides of the invention.
  • the fusion peptide-induced hemolysis is more effectively inhibited by peptides of the invention compared to VIRIP.

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Claims (21)

  1. Peptide mit biologischer Aktivität gegen Infektion durch HIV, die die Aminosäuresequenz
    Z1-LE-X1-IP-X2-X3-X4-P-X5-X6-X7-X8-X9-X10-K-X11-X12-X13-X14-X15-Z2
    aufweisen, wobei
    X1 Lysin, Alanin oder Asparaginsäure ist;
    X2 Cystein, Methionin oder Isoleucin ist;
    X3 Serin, Cystein, Lysin oder Glycin ist;
    X4 Isoleucin, Alanin, Phenylalanin oder Cystein ist;
    X5 Prolin, D-Prolin oder ein substituiertes L- oder D-Prolin ist;
    X6 Cystein oder Glutaminsäure ist;
    X7 eine Aminosäure mit einer hydrophoben oder aromatischen Seitenkette oder Cystein ist;
    X8 eine Aminosäure mit einer hydrophoben oder aromatischen Seitenkette oder Cystein ist;
    X9 eine Aminosäure mit einer aromatischen Seitenkette ist;
    X10 Glycin, Alanin oder Asparagin ist;
    X11 Prolin, Asparaginsäure, Octahydroindolyl-2-carbonsäure oder D-1,2,3,4-Tetrahydroisochinolin-3-carbonsäure;
    X12 Phenylalanin, Alanin, Glycin, Glutaminsäure oder D-1,2,3,4-Tetrahydroisochinolin-3-carbonsäure;
    X13 eine Aminosäure mit einer hydrophoben oder aromatischen Seitenkette ist;
    X14 eine Aminosäure mit einer hydrophoben oder aromatischen Seitenkette ist;
    X15 Phenylalanin oder eine Deletion ist;
    Z1 = NH2 oder eine Sequenz von 1 bis 10 Aminosäureresten ist;
    Z2 = COOH oder eine Sequenz von 1 bis 10 Aminosäureresten ist;
    und Peptide, die kovalent verknüpfte Oligomere und/oder amidierte, alkylierte, acylierte, sulfatierte, PEGylierte, phosphorylierte und/oder glycosylierte Derivate sind;
    mit den folgenden Maßgaben:
    (a) Wenn X12 Alanin, Glycin, Glutaminsäure oder D-1,2,3,4-Tetrahydroisochinolin-3-carbonsäure ist, dann sind X13, X14 und X15 Phenylalanin, Valin bzw. Phenylalanin; und/oder
    (b) wenn X12 Phenylalanin ist, dann sind X13, X14 und X15 Valin, Phenylalanin bzw. eine Deletion; und
    (c) es gibt maximal zwei Cysteinreste in einem Peptid.
  2. Peptide gemäß Anspruch 1 mit einer biologischen Aktivität gegen HIV-Infektion, die die Aminosäuresequenz
    Z1-LE-X1-IP-X2-X3-X4-P-X5-X6-X7-X8-X9-X10-K-X11-FVF-Z2
    aufweisen, wobei
    X1 Lysin, Alanin oder Asparaginsäure ist;
    X2 Cystein, Methionin oder Isoleucin ist;
    X3 Serin, Cystein oder Glycin ist;
    X4 Isoleucin oder Cystein ist;
    X5 Prolin, D-Prolin oder irgendein substituiertes L- oder D-Prolin ist;
    X6 Cystein oder Glutaminsäure ist;
    X7 Phenylalanin, Cystein, Valin, Isoleucin oder 3,3-Diphenylalanin ist;
    X8 Phenylalanin, Leucin, Alanin, Glycin, Cystein, D-1,2,3,4-Tetrahydroisochinolin-3-carbonsäure oder L-1,2,3,4-Tetrahydroisochinolin-3-carbonsäure ist;
    X9 eine Aminosäure mit einer aromatischen Seitenkette ist;
    X10 Glycin oder Asparagin ist;
    X11 Prolin oder D-1,2,3,4-Tetrahydroisochinolin-3-carbonsäure ist;
    Z1 = NH2 oder eine Sequenz von 1 bis 10 Aminosäureresten ist;
    Z2 = COOH oder eine Sequenz von 1 bis 10 Aminosäureresten ist;
    und Peptide, die kovalent verknüpfte Oligomere und/oder amidierte, alkylierte, acylierte, sulfatierte, PEGylierte, phosphorylierte und/oder glycosylierte Derivate sind;
    mit den folgenden Maßgaben:
    (a) Wenn zwei Cysteinreste vorhanden sind, die diese Reste durch vier andere Aminosäurereste voneinander getrennt; und
    (b) wenn L-1,2,3,4-Tetrahydroisochinolin-3-carbonsäure (L-Tic), D-1,2,3,4-Tetrahydroisochinolin-3-carbonsäure (D-Tic) und/oder 3,3-Diphenylalanin vorhanden sind, ist kein Cysteinrest vorhanden.
  3. Peptide gemäß Anspruch 1 oder 2 mit einer biologischen Aktivität gegen HIV-Infektion, die die Aminosäuresequenz
    Z1-LE-X1-IP-X2-X3-IP-X5-X6-X7-X8-F-X10-KPFVF-Z2
    aufweisen, wobei
    X1 Lysin, Alanin oder Asparaginsäure ist;
    X2 Cystein, Methionin oder Isoleucin ist;
    X3 Serin oder Glycin ist;
    X5 L-Prolin, D-Prolin oder irgendein substituiertes L- oder D-Prolin ist;
    X6 Cystein oder Glutaminsäure ist;
    X7 Phenylalanin oder Valin ist;
    X8 Phenylalanin, Leucin, Alanin oder L-1,2,3,4-Tetrahydroisochinolin-3-carbonsäure ist;
    X10 Glycin oder Asparagin ist;
    Z1 = NH2 oder eine Sequenz von 1 bis 10 Aminosäureresten ist;
    Z2 = COOH oder eine Sequenz von 1 bis 10 Aminosäureresten ist; und Peptide, die kovalent verknüpfte Oligomere und/oder amidierte, alkylierte, acylierte, sulfatierte, PEGylierte, phosphorylierte und/oder glycosylierte Derivate sind.
  4. Peptide gemäß Anspruch 1 bis 3, die die Aminosäuresequenz
    Z1-LEAIP-X2-SIP-X5-X6-V-X8-FNKPFVF-Z2
    aufweisen, wobei
    X2 und X6 Cysteine sind oder X2 Methionin ist und X6 Glutaminsäure ist; X5 D-Prolin oder L-Prolin ist;
    X8 eine Aminosäure mit einer hydrophoben oder aromatischen Seitenkette oder Lysin ist;
    Z1 = NH2 oder eine Sequenz von 1 bis 10 Aminosäureresten ist;
    Z2 = COOH oder eine Sequenz von 1 bis 10 Aminosäureresten ist;
    und Peptide, die kovalent verknüpfte Oligomere und/oder amidierte, alkylierte, acylierte, sulfatierte, PEGylierte, phosphorylierte und/oder glycosylierte Derivate sind, mit einer biologischen Aktivität gegen HIV-Infektion, mit der Maßgabe, dass wenigstens eine der folgenden Bedingungen gilt:
    X5 ist D-Prolin, oder
    X8 ist nicht Lysin, oder
    X2 und X6 sind Cystein.
  5. Peptide gemäß einem der Ansprüche 1 bis 4, wobei die Cysteinreste auf den Positionen 6 und 11, 6 und 12, 7 und 12 oder 8 und 13 durch eine intramolekulare Disulfidbindung miteinander verbunden sind.
  6. Peptide gemäß einem der Ansprüche 1 bis 4 mit einem einzigen Cysteinrest, wobei der Cysteinrest durch eine intermolekulare Disulfidbindung mit einem anderen Peptid mit einem einzigen Cysteinrest verbunden ist, wobei ein Homodimer entsteht.
  7. Peptide gemäß einem der Ansprüche 1 bis 6, wobei der Leucinrest auf Aminosäureposition 1 und die Glutaminsäure auf Aminosäureposition 2 durch eine N-alkylierte Amidbindung oder durch eine Esterbindung oder durch eine reduzierte Peptidbindung oder durch eine Retro-Inverso-Peptidbindung oder durch eine N-alkylierte Retro-Inverso-Peptidbindung kovalent miteinander verknüpft sind.
  8. Peptide gemäß einem der Ansprüche 1 bis 7 mit einer der Aminosäuresequenzen: VIR-121 LEAIPMSIPpEVAFNKPFVF SEQ ID Nr. 2 VIR-161 LEAIPCSIPpCVAFNKPFVF SEQ ID Nr. 3 VIR-162 LEAIPCSIPPCVGFGKPFVF SEQ ID Nr. 4 VIR-163 LEAIPCSIPPCVLFNKPFVF SEQ ID Nr. 5 VIR-164 LEAIPCSIPPCVFFNKPFVF SEQ ID Nr. 6 VIR-165 LEAIPCSIPPCFAFNKPFVF SEQ ID Nr. 7 VIR-166 LEAIPCSIPPCVA(D-Tic)NKP(D-Tic)FVF SEQ ID Nr. 8 VIR-170 LEAIPMSIPPEVFFGKPFVF SEQ ID Nr. 9 VIR-175 LEAIPMSIPPEFLFGKPFVF SEQ ID Nr. 10 VIR-182 LEAIPMSIPPELAFAKPFVF SEQ ID Nr. 11 VIR-184 LEAIPMSIPPEIAFNKPFVF SEQ ID Nr. 12 VIR-190 LEAIPMSIPpEVGFGKPFVF SEQ ID Nr. 13 VIR-191 LEAIPMSIPpEVLFGKPFVF SEQ ID Nr. 14 VIR-192 LEAIPMSIPpEVFFGKPFVF SEQ ID Nr. 15 VIR-193 LEAIPMSIPpEFAFNKPFVF SEQ ID Nr. 16 VIR-197 LEAIPMSIPpEVFFNKPFVF SEQ ID Nr. 17 VIR-199 LEAIPMSIPpEFLFNKPFVF SEQ ID Nr. 18 VIR-229 LEAIPISIPpEVAFNKPFVF SEQ ID Nr. 19 VIR-234 LEAIPMGIPpEVAFNKPFVF SEQ ID Nr. 20 VIR-243 LEAIPMSIPPEFAFNKDFVF SEQ ID Nr. 21 VIR-252 LEDIPMSIPpEVAFNKPFVF SEQ ID Nr. 22 VIR-255 LEKIPMSIPpEVAFNKPFVF SEQ ID Nr. 23 VIR-257 LEAIPMSIPpEV(cyclohexylalanin)FNKPFVF SEQ ID Nr. 24 VIR-258 LEAIPMSIPpE(1-naphthylalanin)AFNKPFVF SEQ ID Nr. 25 VIR-259 LEAIPMSIPpE(p-fluorphenylalanin)AFNKPFVF SEQ ID Nr. 26 VIR-260 LEAIPMSIPpEV(4-pyridylalanin)FNKPFVF SEQ ID Nr. 27 VIR-261 LEAIPMSIPpE(3,3-diphenylalanin)AFNKPFVF SEQ ID Nr. 28 VIR-262 LEAIPMSIPpEV(D-Tic)FNKPFVF SEQ ID Nr. 29 VIR-263 LEAIPMSIPpEV(L-Tic)FNKPFVF SEQ ID Nr. 30 VIR-264 LEAIPMSIPpEV(3-benzothienylalanin)FNKPFVF SEQ ID Nr. 31 VIR-265 LEAIPMSIPpEV (3-thienylAlanin) FNKPFVF SEQ ID Nr. 32 VIR-266 LEAIPMSIPpEVWFNKPFVF SEQ ID Nr. 33 VIR-268 LEAIPMSIPpEVAFNK (L-Tic) FVF SEQ ID Nr. 34 VIR-269 LEAIPMSIPpEVAFNK (Oic) FVF SEQ ID Nr. 35 VIR-272 LEAIPMCIPPECLFNKPFVF SEQ ID Nr. 36 VIR-273 LEAIPMCIPPECFFNKPFVF SEQ ID Nr. 37 VIR-274 LEAIPMCIPPECLFGKPFVF SEQ ID Nr. 38 VIR-280 LEAIPCSIPPCFLFGKPFVF SEQ ID Nr. 39 VIR-284 LEAIPISIPPEVFFGKPFVF SEQ ID Nr. 40 VIR-286 LEAIPISIPPELAFAKPFVF SEQ ID Nr. 41 VIR-290 LEAIPISIPpEVFFGKPFVF SEQ ID Nr. 42 VIR-298 LEAIPISIPpEVWFNKPFVF SEQ ID Nr. 43 VIR-320 LEAIPMGIPpEVFFGKPFVF SEQ ID Nr. 44 VIR-322 LEAIPMGIPpEVFFNKPFVF SEQ ID Nr. 45 VIR-323 LEAIPMGIPpEFLFNKPFVF SEQ ID Nr. 46 VIR-326 LEDIPMGIPpEVAFNKPFVF SEQ ID Nr. 47 VIR-328 LEAIPMGIPpEVWFNKPFVF SEQ ID Nr. 48 VIR-344 LEAIPCSIPPCVFFGKPFVF SEQ ID Nr. 49 VIR-345 LEAIPCSIPPCFLFGKPFVF SEQ ID Nr. 50 VIR-346 LEAIPCSIPPCLAFAKPFVF SEQ ID Nr. 51 VIR-348 LEAIPCSIPpCVGFGKPFVF SEQ ID Nr. 52 VIR-350 LEAIPCSIPpCVFFGKPFVF SEQ ID Nr. 53 VIR-351 LEAIPCSIPpCFAFNKPFVF SEQ ID Nr. 54 VIR-352 LEAIPCSIPpCVFFNKPFVF SEQ ID Nr. 55 VIR-353 LEAIPCSIPpCFLFNKPFVF SEQ ID Nr. 56 VIR-354 LEAIPCSIPpCVAFNKPFVF SEQ ID Nr. 57 VIR-355 LEAIPCGIPpCVAFNKPFVF SEQ ID Nr. 58 VIR-356 LEAIPCSIPPCFAFNKDFVF SEQ ID Nr. 59 VIR-357 LEDIPCSIPpCVAFNKPFVF SEQ ID Nr. 60 VIR-358 LEKIPCSIPpCVAFNKPFVF SEQ ID Nr. 61 VIR-376 LEAIPMSIPpEFLFGKPAFVF SEQ ID Nr. 62 VIR-377 LEAIPMSIPpEFLFGKPGFVF SEQ ID Nr. 63 VIR-380 LEAIPMSIPpEFLFGKPFFVF SEQ ID Nr. 64 VIR-384 LEAIPMSIPpEFLFGKPEFVF SEQ ID Nr. 65 VIR-396 LEAIPMSAPpEFLFGKPFVF SEQ ID Nr. 66 VIR-400 LEAIPMSFPpEFLFGKPFVF SEQ ID Nr. 67 VIR-416 LEAIPMGIPpEFLFGKPFVF SEQ ID Nr. 68 VIR-418 LEKIPMGIPpEFLFGKPFVF SEQ ID Nr. 69 VIR-445 LEAIPISIPpEV(D-Tic)FNKPFVF SEQ ID Nr. 70 VIR-447 LEAIPISIPpEVAFNK(L-Tic)FVF SEQ ID Nr. 71 VIR-448 LEAIPMGIPpEV(D-Tic)FNKPFVF SEQ ID Nr. 72 VIR-449 LEAIPMGIPpEV(L-Tic)FNKPFVF SEQ ID Nr. 73 VIR-452 LEDIPMSIPpEV(L-Tic)FNKPFVF SEQ ID Nr. 74 VIR-454 LEKIPMSIPpEV(D-Tic)FNKPFVF SEQ ID Nr. 75 VIR-455 LEKIPMSIPpEV(L-Tic)FNKPFVF SEQ ID Nr. 76 VIR-479 LEDIPIGIPpEFLFNKPFVF SEQ ID Nr. 77 VIR-483 LEKIPIGIPpEV(D-Tic)FNKPFVF SEQ ID Nr. 78 VIR-484 LEKIPIGIPpEV(L-Tic)FNKPFVF SEQ ID Nr. 79 VIR-485 LEKIPIGIPpEVAFNK(L-Tic)FVF SEQ ID Nr. 80 VIR-487 LEDIPIGIPpEV(L-Tic)FNKPFVF SEQ ID Nr. 81 VIR-488 LEDIPIGIPpEVAFNK(L-Tic)FVF SEQ ID Nr. 82 VIR-512 N-Me-LEAIPMSIPPEFLFGKPFVF SEQ ID Nr. 83 VIR-568 LEAIPMSCPPEFCFGKPFVF SEQ ID Nr. 84 VIR-570 LEAIPCSIPPECLFGKPFVF SEQ ID Nr. 85 VIR-576 (LEAIPCSIPPEFLFGKPFVF)2 SEQ ID Nr. 86 VIR-580 LEAIPMSIPPEFLFGKPFVF-miniPEG SEQ ID Nr. 87 VIR-590 LEAIPMKIPPEFLFGKPFVF SEQ ID Nr. 88.
  9. Peptide gemäß einem der Ansprüche 1 bis 8, die mit dem Fusionspeptid von HIV wechselwirken.
  10. Peptide gemäß einem der Ansprüche 1 bis 9, die ein gemäß Beispiel 3 gemessenes IC50 von kleiner oder gleich 6500 nM in Bezug auf die Hemmung des X4-tropen HIV-1-NL-4-3 aufweisen, vorzugsweise solche, die ein IC50 von kleiner oder gleich 2000 nM aufweisen, und am meisten bevorzugt solche, die ein IC50 von kleiner oder gleich 800 nM aufweisen, wie VIR-344 (SEQ ID Nr. 49) mit einem IC50 von 348 nM, VIR-345 (SEQ ID Nr. 50) mit einem IC50 von 298 nM, VIR-353 (SEQ ID Nr. 56) mit einem IC50 von 225 nM, VIR-357 (SEQ ID Nr. 60) mit einem IC50 von 497 nM, VIR-358 (SEQ ID Nr. 61) mit einem IC50 von 706 nM, VIR-449 (SEQ ID Nr. 73) mit einem IC50 von 274 nM, VIR-455 (SEQ ID Nr. 76) mit einem IC50 von 134 nM, VIR-484 (SEQ ID Nr. 79) mit einem IC50 von 100 nM, VIR-512 (SEQ ID Nr. 83) mit einem IC50 von 138 nM, VIR-576 (SEQ ID Nr. 86) mit einem IC50 von 107 nM und VIR-580 (SEQ ID Nr. 87) mit einem IC50 von 150 nM.
  11. Nucleinsäuren, die für Peptide gemäß einem der Ansprüche 1 bis 10 codieren.
  12. Antikörper, die spezifisch an Peptide gemäß den Ansprüchen 1 bis 10 binden.
  13. Medikament, das die Peptide gemäß den Ansprüchen 1 bis 10, Nucleinsäuren gemäß Anspruch 11 oder Antikörper gemäß Anspruch 12 enthält.
  14. Medikament gemäß Anspruch 13 in galenischen Zubereitungen zur oralen, intravenösen, intramuskulären, intrakutanen, subkutanen, intrathekalen Verabreichung und als Aerosol zur transpulmonalen Verabreichung.
  15. Medikament gemäß Anspruch 13 oder 14, das wenigstens ein weiteres Therapeutikum umfasst.
  16. Medikament gemäß Anspruch 15, wobei das wenigstens eine weitere Therapeutikum ein Inhibitor der viralen Protease, ein Inhibitor der Reversen Transcriptase, ein Fusionsinhibitor, ein Cytokin, ein Cytokininhibitor, ein Glycosylierungsinhibitor oder ein Inhibitor der viralen mRNA ist.
  17. Verwendung der Peptide gemäß den Ansprüchen 1 bis 10 zur Herstellung eines Medikaments zur Behandlung von HIV-Infektionen.
  18. Assay zur Bestimmung von Molekülen, die zur Wechselwirkung mit dem Fusionspeptid von HIV befähigt sind, umfassend ein Peptid gemäß einem der Ansprüche 1 bis 10, wobei der Assay nicht am Menschen durchgeführt wird.
  19. Verwendung der Peptide gemäß einem der Ansprüche 1 bis 10 in einem Assay gemäß Anspruch 18.
  20. Diagnostisches Mittel, das Peptide gemäß einem der Ansprüche 1 bis 10, Nucleinsäuren gemäß Anspruch 11 oder Antikörper gemäß Anspruch 12 enthält.
  21. Verwendung des diagnostischen Mittels gemäß Anspruch 18 für Assaysysteme zum Testen von isoliertem Plasma, Gewebe, Urin und Liquor auf das Niveau der HIV-Infektion.
EP03813584A 2002-12-19 2003-12-19 Peptide und ihre verwendung zur behandlung von hiv infektionen Expired - Lifetime EP1572743B1 (de)

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